For over a century since the discovery of X-rays by Roentgen, students in radiological sciences have been taught that the quintessential target for radiation induced genetic damages resides in the DNA of the nucleus. The biological consequences of cytoplasmic damage are largely unknown. Using a precision charged particle microbeam, the applicant and his co-investigators have previously shown that targeted cytoplasmic irradiation induces mutations in the nucleus of the same hit cell in a process involving reactive oxygen and nitrogen species. Recent preliminary data suggest that the consequence of these damages can result in a non-targeted/ bystander response and that progeny of hit cells show an increase in genomic instability. The overall goals of this competitive renewal application are i) to characterize the induction of genomic instability in a human small airway epithelial model induced by cytoplasmic irradiation; 2) to define the role of mitochondrial alterations (fusion/ fission) and the subsequent functional alterations induced by cytoplasmic irradiation; and 3) to determine signaling pathways induced by cytoplasmic irradiation that result in the biological response. A series of three interrelated specific aims are proposed to elucidate three testable hypotheses; linking the three themes of the application: genomic instability, mitochondrial damage and inflammatory signaling pathways. A state of the art charge particle microbeam that can deliver a precise number of alpha particles with a beam size of ~ 1 micrometer and a precision greater than 98% provides an unprecedented opportunity to address an issue that has been of interest to radiobiologists for decades: the differential biological effects of nuclear versus cytoplasmic damage. A major paradigm shift in radiation biology in the last decade has resulted from the elucidation of the biological consequence of targeted cytoplasmic irradiation and discovery of the bystander effect. Together with the genomic instability and bystander effects, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells. Such studies are critically important understanding the cellular response to DNA damages and in low dose radiation risk assessment.